Visualization of X chromosome reactivation in mouse primordial germ cells in vivo

ABSTRACT X chromosome inactivation (XCI), determined during development, remains stable after embryonic cell divisions. However, primordial germ cells (PGCs) are exceptions in that XCI is reprogrammed and inactivated X chromosomes are reactivated. Although interactions between PGCs and somatic cells are thought to be important for PGC development, little is known about them. Here, we performed imaging of X chromosome reactivation (XCR) using the ‘Momiji’ mouse system, which can monitor the X chromosome's inactive and active states using two color fluorescence reporter genes, and investigated whether interactions would affect XCR in PGCs. Based on their expression levels, we found that XCR of the Pgk1 locus began at embryonic day (E)10.5 and was almost complete by E13.5. During this period, PGCs became distributed uniformly in the genital ridge, proliferated, and formed clusters; XCR progressed accordingly. In addition, XCR of the Pgk1 locus preceded that of the Hprt locus, indicating that the timing of epigenetic memory erasure varied according to the locus of each of these X-linked genes. Our results indicate that XCR proceeds along with the proliferation of PGCs clustered within the genital ridge. This article has an associated First Person interview with the first author of the paper.

Comparative analyses of gene expression in common marmoset and human pluripotent stem cells (PSCs) identify factors enhancing homologous recombination efficiency in the HPRT locus of human PSCs

A previous study assessing the efficiency of the genome editing technology CRISPR-Cas9 for knock-in gene targeting in common marmoset (marmoset; Callithrix jacchus) embryonic stem cells (ESCs) unexpectedly identified innately enhanced homologous recombination (HR) activity in marmoset ESCs (cmESCs). Here, we compared gene expression in marmoset and human pluripotent stem cells (PSCs) using transcriptomic and quantitative PCR (qPCR) analyses and found that five HR-related genes (BRCA1, BRCA2, RAD51C, RAD51D and RAD51) were upregulated in marmoset cells. Four of these upregulated genes enhanced HR efficiency with CRISPR-Cas9 in human pluripotent stem cells. Thus, the present study provides a novel insight into species-specific mechanisms for the choice of DNA repair pathways.

Integrin-mediated axoglial interactions initiate myelination in the central nervous system

All but the smallest-diameter axons in the central nervous system are myelinated, but the signals that initiate myelination are unknown. Our prior work has shown that integrin signaling forms part of the cell–cell interactions that ensure only those oligodendrocytes contacting axons survive. Here, therefore, we have asked whether integrins regulate the interactions that lead to myelination. Using homologous recombination to insert a single-copy transgene into the hypoxanthine phosphoribosyl transferase (hprt) locus, we find that mice expressing a dominant-negative β1 integrin in myelinating oligodendrocytes require a larger axon diameter to initiate timely myelination. Mice with a conditional deletion of focal adhesion kinase (a signaling molecule activated by integrins) exhibit a similar phenotype. Conversely, transgenic mice expressing dominant-negative β3 integrin in oligodendrocytes display no myelination abnormalities. We conclude that β1 integrin plays a key role in the axoglial interactions that sense axon size and initiate myelination, such that loss of integrin signaling leads to a delay in myelination of small-diameter axons.

Targeted transgenesis at the HPRT locus: an efficient strategy to achieve tightly controlled in vivo conditional expression with the tet system

The tet-inducible system has been widely used to achieve conditional gene expression in genetically modified mice. To alleviate the frequent difficulties associated with recovery of relevant transgenic founders, we tested whether a controlled strategy of transgenesis would support reliable cell-specific, doxycycline (Dox)-controlled transgene expression in vivo. Taking advantage of the potent hypoxanthine-aminopterin-thymidine selection strategy and an embryonic stem (ES) cell line supporting efficient germ-line transmission, we used hypoxanthine phosphoribosyltransferase ( HPRT) targeting to insert a single copy tet-inducible construct designed to allow both glucocorticoid receptor (GR) and β-galactosidase (β-Gal) expression. Conditional, Dox-dependent GR and β-Gal expression was evidenced in targeted ES cells. Breeding ES-derived single copy transgenic mice with mice bearing appropriate tet transactivators resulted in β-Gal expression both qualitatively and quantitatively similar to that observed in mice with random integration of the same construct. Interestingly, GR expression in mice was dependent on transgene orientation in the HPRT locus while embryonic stem cell expression was not. Thus, a conditional construct inserted in single copy and in predetermined orientation at the HPRT locus demonstrated a Dox-dependent gene expression phenotype in adult mice suggesting that controlled insertion of tet-inducible constructs at the HPRT locus can provide an efficient alternative strategy to reproducibly generate animal models with tetracycline-induced transgene expression.

A GABP-binding element in the Robo4 promoter is necessary for endothelial expression in vivo

We recently demonstrated that the 3-kb 5′-flanking region of the human ROBO4 gene directs endothelial cell–specific expression in vitro and in vivo. Moreover, a GA-binding protein (GABP)–binding motif at −119 was necessary for mediating promoter activity in vitro. The goal of the present study was to confirm the functional relevance of the −119 GABP-binding site in vivo. To that end, the Hprt locus of mice was targeted with a Robo4-LacZ transgenic cassette in which the GABP site was mutated. In other studies, the GABP mutation was introduced into the endogenous mouse Robo4 locus in which LacZ was knocked-in. Compared with their respective controls, the mutant promoters displayed a significant reduction in activity in embryoid bodies, embryos, and adult animals. Together, these data provide strong support for the role of the GABP-binding motif in mediating Robo4 expression in the intact endothelium.